RESUMO
Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malabsorption, which induces severe neuro-ophthalmic complications. Currently, treatment combines a low-fat diet with high-dose vitamin A and E supplementation but still fails in normalizing serum vitamin E levels and providing complete ophthalmic protection. To explore these persistent complications, we developed two knock-out cell models of FHBL-SD1 and FHBL-SD3 using the CRISPR/Cas9 technique in Caco-2/TC7 cells. DNA sequencing, RNA quantification and Western blotting confirmed the introduction of mutations with protein knock-out in four clones associated with i) impaired lipid droplet formation and ii) defective triglyceride (-57.0 ± 2.6% to -83.9 ± 1.6%) and cholesterol (-35.3 ± 4.4% to -60.6 ± 3.5%) secretion. A significant decrease in α-tocopherol secretion was also observed in these clones (-41.5 ± 3.7% to -97.2 ± 2.8%), even with the pharmaceutical forms of vitamin E: tocopherol-acetate and tocofersolan (α-tocopheryl polyethylene glycol succinate 1000). MTTP silencing led to a more severe phenotype than SAR1B silencing, which is consistent with clinical observations. Our cellular models thus provide an efficient tool to experiment with therapeutic strategies and will allow progress in understanding the mechanisms involved in lipid metabolism.
Assuntos
Hipobetalipoproteinemias , Proteínas Monoméricas de Ligação ao GTP , Humanos , alfa-Tocoferol , Apolipoproteínas B/genética , Células CACO-2 , Enterócitos/metabolismo , Hipobetalipoproteinemias/genética , Hipobetalipoproteinemias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Vitamina E/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Citrullus colocynthis (L.) Schrad is a common fruit in traditional medicine and used as remedy against various diseases, especially diabetes. Up to now, its anti-diabetic effects have been fully attributed to its enhancement of pancreatic insulin secretion. Whether C. colocynthis also ameliorates insulin action in peripheral tissues has not been investigated. AIM OF THE STUDY: In the present study, using 3T3-L1 adipocytes as cell model, we have investigated whether colocynth fruit extracts affect insulin action. MATERIALS AND METHODS: Various extracts were prepared from the C. colocynthis fruit and screened using a cell-based 96 well plate GLUT4 translocation assay. Promising extracts were further studied for their effects on glucose uptake and cell viability. The effect on insulin signal transduction was determined by Western blot and the molecular composition was established by LC-MS. RESULTS: The ethyl acetate fractions of aqueous non-defatted extracts of seed and pulp, designated Sna1 and Pna1, acutely enhanced insulin-induced GLUT4 translocation. In accordance, both extracts increased insulin-stimulated cellular glucose uptake. Pna1, which displayed greater effects on GLUT4 and glucose uptake than Sna1, was further investigated and was demonstrated to increase GLUT4 translocation without changing the half-maximum dose (ED50) of insulin, nor changing GLUT4 translocation kinetics. At the molecular level, Pna1 was found to enhance insulin-induced PKB phosphorylation without changing phosphorylation of the insulin receptor. Pna1 appeared not to be toxic to cells and, like insulin, restored cell viability during serum starvation. By investigating the molecular composition of Pna1, nine compounds were identified that made up 87% of the mass of the extract, one of which is likely to be responsible for the insulin-enhancing effects of Pna1. CONCLUSIONS: The C. colocynthis fruit possesses insulin-enhancing activity. This activity may explain in part its anti-diabetic effects in traditional medicine. It also identifies the C. colocynthis as a source of a potential novel insulin enhancer that may prove to be useful to reduce hyperglycemia in type 2 diabetes.
Assuntos
Citrullus colocynthis/química , Frutas/química , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes/farmacologia , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/química , Insulina/metabolismo , Resistência à Insulina , Medicina Tradicional , Camundongos , Fosforilação/efeitos dos fármacos , Extratos Vegetais/química , Transporte ProteicoRESUMO
Thermogenic brown and brite adipocytes convert chemical energy from nutrients into heat. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to control fat mass such as in obesity or cachexia. The peroxisome proliferator-activated receptor (PPAR) family plays key roles in the maintenance of adipose tissue and in the regulation of thermogenic activity. Activation of these receptors induce browning of white adipocyte. The purpose of this work was to characterize the role of carnosic acid (CA), a compound used in traditional medicine, in the control of brown/brite adipocyte formation and function. We used human multipotent adipose-derived stem (hMADS) cells differentiated into white or brite adipocytes. The expression of key marker genes was determined using RT-qPCR and western blotting. We show here that CA inhibits the browning of white adipocytes and favors decreased gene expression of thermogenic markers. CA treatment does not affect ß-adrenergic response. Importantly, the effects of CA are fully reversible. We used transactivation assays to show that CA has a PPARα/γ antagonistic action. Our data pinpoint CA as a drug able to control PPAR activity through an antagonistic effect. These observations shed some light on the development of natural PPAR antagonists and their potential effects on thermogenic response.
Assuntos
Abietanos/farmacologia , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Rosmarinus/química , Adipócitos Bege/efeitos dos fármacos , Adipócitos Bege/metabolismo , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Lipólise/efeitos dos fármacos , Camundongos , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Rosiglitazona/farmacologia , Termogênese/efeitos dos fármacos , Termogênese/genéticaRESUMO
PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.